Premature infants admitted to the neonatal intensive care unit (NICU) require up to several hundred procedures during their hospitalization. Many of these are tissue damaging procedures (TDPs) that cause pain. NIH-funded neonatal pain research currently focuses primarily on the relationship between pain, analgesia, and neurodevelopmental outcomes. However, recent multicenter clinical studies aimed at improving neurologic outcome by decreasing pain with morphine were inconclusive [Bellu et al, 2008;[Anand et al, 2004;Simons et al, 2003]. They suggest that prevention and treatment of pain alone may not significantly decrease neurologic injury. Although TDPs are known to cause hypoxemia (reduced arterial oxygen content), it is not known whether they result in hypoxia (oxygen deficiency at the tissue level), which leads to oxidative stress and cell injury. Answering this research question will fill this important gap in neonatal pain research while complementing the other projects in NIH's neonatal pain portfolio. The answer will make possible the development of safer, more effective clinical interventions to improve health outcomes for premature infants cared for in NICUs worldwide (our long-term goal). This RO1 application from a new investigator will measure the effects of two of the most commonly performed NICU procedures representative of TDPs-endotracheal suctioning and heel lance in a randomized prospective clinical trial of premature neonates. Endotracheal suctioning refers to the removal of mucus or secretions from the endotracheal tube using an in-line catheter without disconnection from the ventilator. Heel lance refers to the puncture of a newborn's heel for blood glucose using a specially designed lancet. Premature infants experiencing heel lance will be randomized into either a placebo or sucrose intervention group. Blood sampling for biochemical measurement of hypoxia, oxidative stress and cell injury will be timed before and after the procedures (to be performed as clinically indicated). Our general hypothesis is that commonly performed tissue damaging procedures exhibit a positive correlation between biobehavioral markers of pain and biochemical markers of hypoxia, oxidative stress, and cell injury. Specific Aim 1 will determine whether endotracheal suctioning and/or heel lance with and without sucrose intervention) increase biobehavioral markers of pain. Pain will be quantified using a validated pain scoring tool, the Premature Infant Pain Profile (PIPP). Specific Aim 2 will determine whether endotracheal suctioning and/or heel lance with and without sucrose intervention) increase biochemical markers of hypoxia and oxidative stress. Products of ATP breakdown in plasma-hypoxanthine (Hx), xanthine (Xa), and uric acid (UA)-will be measured using high performance liquid chromatography. Oxidative stress will be quantified by measuring plasma levels of xanthine oxidase (XO), xanthine dehydrogenase (XDH), and allantoin using gas chromatography and mass spectroscopy. Specific Aim 3 will determine whether endotracheal suctioning and/or heel lance with and without sucrose intervention) increase biochemical markers of cell injury Cell injury will be quantified by measuring plasma levels of malondialdehyde (MDA), a marker for lipid peroxidation. Specific Aim 4 will determine whether there is a positive correlation between biobehavioral markers of procedural pain and biochemical markers of hypoxia, oxidative stress, and cell injury. PUBLIC HEALTH RELEVANCE: Procedural pain in premature infants and its relationship to hypoxia (oxygen deficiency) is an important issue for neonatal intensive care practice as well as long-term infant neurodevelopment. If early pain experiences are shown to contribute to hypoxia, oxidative stress, and cell injury, biochemical markers might be used to provide biochemical links between the cellular consequences of early pain and its neurologic consequences. These biomarkers may then be used to evaluate mechanism-based interventions that could lead to more effective management of NICU procedural pain and long-term infant neurodevelopment.